JP2007228905A - Method for detecting protein phosphatase activity on chip - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for profiling especially kinetics in various protein phosphatases comprehensively by detecting the phosphorylation of substrate peptide by a simple procedures rapidly by using an inexpensive substance and without requiring a specific technology. <P>SOLUTION: This method for detecting the protein phosphatase activity by detecting the phosphorylation of the peptide or protein immobilized on a substrate plate is characterized by effecting e.g. a chelating compound described in formula (I) on the phosphorilated peptide or protein, and then detecting the de-phosphorylation by using a fluorescence or chemical luminescence. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for analyzing protein phosphatase activity using an array in which one or more kinds of peptides or proteins are immobilized on a substrate, wherein a chelating compound acts on the peptide or protein phosphorylated on the array And detecting the protein phosphatase activity by detection using fluorescence or chemiluminescence. According to the present invention, it is possible to realize a rapid and efficient analysis of protein phosphatase activity that is simple and comprehensive.

  In recent years, research on intracellular signal transduction has progressed dramatically, not only how signals are transmitted to the nucleus from cell surface receptors activated by growth factors and cytokines, but also cell cycle, adhesion, The reality of various signaling pathways that control movement, polarity, morphogenesis, differentiation, life and death, etc. has become clear. These signaling pathways do not function independently, but cross-talk with each other and function as a system. The causes of various diseases including cancer have been explained as abnormalities in these signal transduction pathways.

  In the signal transduction pathway described above, it is known that various types of protein kinases involved in protein phosphorylation modification play an important role in a complex manner. At the same time, protein phosphatase, which catalyzes the dephosphorylation of phosphorylated protein, occupies an important position. If we can comprehensively analyze the activities of these protein phosphatases and profile their intracellular dynamics at once, we will be able to apply them not only to basic research in cell biology and pharmacology, but also in fields such as drug development and clinical applications. It is expected to contribute greatly. However, at present, a technology that can simultaneously and efficiently profile the dynamics of various protein phosphatases has not yet been established.

For example, a method for confirming protein phosphatase activity by fluorescence resonance energy transfer (FRET) using a substrate peptide of protein phosphatase having a fluorescent moiety has been reported (Patent Document 1). Moreover, the method of analyzing a phosphorylation site | part is shown by measuring a mass spectrum about the signal derived from a phosphorylated peptide (nonpatent literature 1). Furthermore, a method using ³² P has been performed for a long time (Non-patent Document 2). However, these methods have low throughput and are not sufficient for comprehensive analysis.

JP-T-2005-538164 Rapid Commun. Mass Spectrum. , Volume 17, pages 1493-1496 (issued in 2003) Protein kinases and phosphatases (published by Medical Science International, Inc.) pp. 172-188

  The present invention can comprehensively profile the kinetics of various protein phosphatases in particular by detecting dephosphorylation of peptides or proteins by a simple method that uses inexpensive materials and does not require special techniques. Try to establish a method.

  As a result of intensive studies in view of the above circumstances, the present inventors have used chelating compounds to phosphorylated peptides or proteins using an array in which peptides or proteins recognizing protein kinases are immobilized on a substrate. The present inventors have found that a method for detecting phosphorylation using fluorescence or chemiluminescence is extremely useful for rapid and particularly comprehensive analysis of various protein kinase kinetics.

The present invention has the following configuration.
(1) A method for detecting dephosphorylation of a peptide or protein immobilized on a substrate, wherein a chelating compound is allowed to act on the substrate on which the peptide or protein is immobilized, followed by fluorescence or chemiluminescence. A method for detecting protein phosphatase activity, characterized by using the method to detect phosphorylation.
(2) The method for detecting protein phosphatase activity according to (1), wherein the chelating compound is a polyamine zinc complex.
(3) The method for detecting protein phosphatase activity according to (1) or (2), wherein the chelating compound is a binuclear zinc complex having a polyamine compound as a ligand.
(4) The method for detecting protein phosphatase activity according to any one of (1) to (3), wherein the chelating compound is a compound described in formula (I).
(5) The method for detecting protein phosphatase activity according to any one of (1) to (4), wherein the substrate is glass.
(6) The method for detecting protein phosphatase activity according to any one of (1) to (5), wherein the surface is gold.
(7) The method for detecting protein phosphatase activity according to any one of (1) to (6), wherein a peptide having a cysteine residue added thereto is immobilized.
(8) The method for detecting protein phosphatase activity according to any one of (1) to (7), wherein the chelating compound is a compound modified with a fluorescent substance or a chemiluminescent substance.
(9) Any one of (1) to (8), wherein the chelating compound is a compound modified with biotin and avidin or streptavidin modified with a fluorescent substance or a chemiluminescent substance is allowed to act A method for detecting protein phosphatase activity according to claim 1.
(10) A complex of a chelating compound modified with biotin and an avidin or streptavidin modified with a fluorescent substance or a chemiluminescent substance, wherein any one of (1) to (8) A method for detecting protein phosphatase activity.

  The method of the present invention has made it possible to analyze the dynamics of various protein phosphatases in a very simple and rapid manner without requiring special techniques. By using a chelate compound, it is inexpensive and easy to handle, and has a significant advantage over conventional methods in that it is not dependent on the type of phosphorylated amino acid or the amino acid sequence in the vicinity thereof. By using the present invention, it is possible to comprehensively analyze various types of protein phosphatase signals, and effectively profile protein phosphatase kinetics in cells accompanying the introduction of genes with unknown functions or drug administration. be able to. This is expected to expand into genomic drug discovery, such as functional analysis from new genes and approaches to drug discovery.

  The method for detecting a phosphorylated peptide or protein on a substrate in the present invention is performed using a fluorescent substance or a chemiluminescent substance as is well known. The fluorescent substance is not particularly limited, and any fluorescent compound is applied. For example, FITC, 6-FAM. Examples thereof include those used in general fluorescence assays such as Cy3, Cy5, Texas Red, TAMRA, and APC. Moreover, it is not specifically limited about the case where a chemiluminescent substance is used, For example, common compounds, such as a luminol, a luciferin, and a lophine, are illustrated.

  In the present invention, since the peptide or protein used as a substrate is intended to comprehensively profile the kinetics of protein phosphatase, one type of peptide or protein is dephosphorylated only by one type of protein phosphatase, It is ideal and preferred that protein phosphatase is not dephosphorylated. The amino acid sequence serving as a substrate for protein phosphatase is known or can be appropriately selected based on the known sequence. If the array of the present invention immobilizes peptides or proteins of a type corresponding to a plurality of types of protein phosphatases that need to grasp their kinetics, all the protein phosphatases can be profiled with one array. preferable. Of course, a peptide or protein corresponding to only one type of protein phosphatase may be immobilized on one array, and protein phosphatase profiling may be performed using the required number of arrays.

  As the substrate of the array used in the present invention, glass is the most common and preferred, but plastics such as polyethylene terephthalate (PET), polycarbonate, and acrylic can also be mentioned. Transparent glass is easily available and is particularly advantageous in terms of versatility. When a glass substrate is used, the type of glass and the thickness of the substrate are not particularly limited, but the thickness is preferably about 0.1 to 20 mm, for example, a substrate of about 1 to 2 mm is used. Although the size and shape are not particularly limited, it is preferable to use the same size as that of a commercially available slide glass in consideration of compatibility with a widely used array analyzer.

  With respect to the substrate surface, for example, a glass surface that has been subjected to chemical modification such as silane coupling based on a known method and introduced with a functional group can be used. Specific functional groups include an amino group and a carboxyl group. Examples include thiol groups and epoxy groups. Alternatively, the surface of the substrate may be coated with a thin metal layer. Examples of the type of metal to be coated include gold, silver, platinum and the like, but gold that is very stable in acid, alkali, organic solvent, and the like is preferable. In the case of using gold, since there have been many reports and findings on the modification of surface chemistry by using an alkanethiol compound having a functional group at the end as described above, This is useful in that a functional group can be easily introduced to the surface.

  In the case of coating a metal, particularly gold, the method is not particularly limited, but generally a vapor deposition method, a sputtering method, an ion coating method or the like is selected. The thickness of the thin metal layer is preferably controlled at the nano level. The thickness of the metal thin layer is not particularly limited, but is generally selected in the range of 20 to 100 nm, preferably 30 to 80 nm. In order to suppress the peeling of the thin metal layer, a chromium layer or a titanium layer of about 0.5 to 10 nm, preferably about 1 to 5 nm may be coated on the substrate in advance.

  In the present invention, the peptide or protein refers to all ordinary polypeptides in which a plurality of amino acids are linked by condensation bonding. Examples of protein phosphatases to be profiled include enzymes that dephosphorylate amino acids such as serine, threonine, and tyrosine of phosphorylated proteins. For example, PP1, PP2A, PP2B, PP2C, PTPβ, RPTP-BK, SHP -2, TC-PTP, etc. can be exemplified, but basically, it can be applied to all types of protein phosphatases.

  The peptide or protein serving as a substrate for the protein phosphatase may be a known one, but the amino acid sequence can be appropriately selected based on the known sequence. In the case of using a peptide, a length of 100 amino acid residues or less is generally used, preferably 30 amino acid residues, more preferably 8-25 amino acid residues, still more preferably about 10-20 amino acid residues. Is used. Moreover, the molecular weight in the case of using protein is not specifically limited. The method for producing the peptide or protein is not particularly limited, and can be produced by a known chemical or genetic engineering method. In the present invention, the peptide or protein serving as a protein phosphatase substrate refers to a peptide or protein having a property of undergoing a dephosphorylation reaction by the protein phosphatase.

  In the present invention, an array in which peptides or proteins are immobilized on a substrate as described above is used. As the peptide or protein, at least one containing an amino acid sequence capable of functioning as a protein phosphatase substrate, preferably a plurality of amino acid sequences that are dephosphorylated by different types of protein phosphatases are used. Furthermore, it is preferable that the negative control is also immobilized on the same substrate. In terms of ease of synthesis, ease of handling, storage stability, etc., it is more preferable to use a peptide having a relatively low molecular weight.

  The method for immobilizing peptides or proteins to the substrate in the array used in the present invention is not particularly limited, and is a method through amino groups or thiol groups present in polypeptide sequences, or His tag, GST tag, etc. There are various modes such as a method using.

  J. et al. M.M. See by Brockman et al. Am. Chem. Soc. Based on the method reported in Volume 121, pages 8044-8051 (1999), a method of immobilizing via an alkanethiol layer and modifying the background with PEG (polyethylene glycol) may be used. Good. In addition, in order to further suppress non-specific effects, it is also possible to immobilize a peptide after binding a derivative in which a functional group as described above is introduced to the end of PEG to alkanethiol, which also has a spacer effect. It is useful in.

  Specifically, for example, carboxymethyl dextran or a water-soluble polymer such as PEG having a terminal modified with a carboxyl group is bonded to a metal thin film to introduce a carboxyl group on the surface, and EDC (1-ethyl-3, Examples of NHS (N-hydroxysuccinimide) esters using water-soluble carbodiimides such as 4-dimethylaminopropylcarbodiimide) include reacting amino groups of peptides or proteins with activated carboxyl groups. Alternatively, the surface may be modified with maleimide and then immobilized via an amino acid residue containing a thiol group such as cysteine. In this case, the cysteine residue is preferably added to one end of the peptide. In order to reduce the non-specific influence, the latter immobilization method via a thiol group is more preferable, but it is not particularly limited.

  A method of immobilizing a peptide to which a tag such as His-tag or GST described above is added is also very simple and useful. In this case, it is preferable to introduce NTA (nitrilotriacetic acid) and glutathione on the metal thin film after introducing an amino group or a carboxyl group to the metal surface via alkanethiol as described above. In the case of His-tag, the substrate is immobilized after the NTA-introduced array is treated with nickel chloride.

  Among the methods described above, the method of immobilizing a peptide via a thiol group is particularly preferable from the viewpoints of specificity and sensitivity. In this case, the immobilization is preferably performed via a cysteine residue.

  It is preferable that at least one cysteine residue is present in the amino acid sequence of the peptide to be immobilized. The cysteine residue is an amino acid necessary for the peptide to be immobilized to have its original function, even if it exists as an essential residue as an amino acid sequence necessary for the peptide to function. It may be a case where it is further added to the sequence. The position of the cysteine residue in the peptide to be immobilized is not particularly limited, but it is preferably added to at least one end, more preferably only to one end. When a cysteine residue is added to one end, only a cysteine residue may be added, but a spacer is used in order to increase the efficiency of interaction with a substance that acts by increasing the degree of freedom of the immobilized peptide. As an amino acid sequence of 1 to several residues may be further added. The amino acid sequence of the spacer portion is not particularly limited, but it is particularly preferable to add a sequence having 1 to several glycine residues and / or alanine residues or serine residues. Alternatively, a hydrophilic polymer such as PEG (polyethylene glycol) may be inserted between the terminal cysteine residue and the substrate sequence instead of the amino acid sequence as described above.

  As described above, when a peptide is immobilized via a thiol group, it is preferable to introduce a heterobifunctional linker. The chemical structure of the heterobifunctional group type linker is not particularly limited, but after introducing an amino group on the surface in advance, a heterobifunctional type having a succinimide (NHS) group or a succinimide sulfate group and a maleimide (MAL) group It is particularly preferable to use a crosslinking agent. As such a heterobifunctional type crosslinking agent, it is possible to use a hydrophilic polymer such as polyethylene glycol (PEG) in which both ends are modified with NHS groups and MAL groups. In order to improve the yield, a lower molecular weight may be used. Specifically, the compound Succinimidyl 4- [N-maleimidomethyl] 4- [N-maleimidomethyl] cyclohexane-1-carboxylate (hereinafter referred to as SMCC) or the compound Sulfosuccinimidyl 4- [N-maleimidecylimide] represented by the formula (III) -1-carboxylate (hereinafter referred to as SSMCC). It should be noted that not only compounds having the same structure as the compound represented by formula (II) or formula (III) but also compounds analogized within a range not impairing the function thereof are also included. In the present invention, both SMCC and SSMCC can be applied. However, from the viewpoint of solubility in water, it is more preferable to use SSMCC when the reaction is carried out in an aqueous system such as a buffer solution.

  When using a polymer having both ends of a hydrophilic polymer such as PEG modified with NHS groups and MAL groups, the size is not particularly limited. Preferably, for example, the molecular weight is preferably about 500 to 5,000, more preferably about 800 to 2,000, and still more preferably about 600 to 1200.

  As described above, a peptide or protein solution is allowed to act on a substrate having a functional group introduced on the surface to produce an array. In this case, it is possible to carry out the manual operation, but such as an automatic spotter. It is preferable to use a spotting device. The type and type of the automatic spotter is not particularly limited, and a spotting device that is generally popular can be used. Although it does not specifically limit about the magnitude | size of a spot, About 1-1000 micrometers is preferable, 10-600 micrometers is more preferable, 100-500 micrometers is still more preferable. The peptide or protein solution to be spotted is preferably a solution dissolved in a buffer solution such as PBS, but an organic solvent such as DMSO (dimethyl sulfoxide) may be mixed in an appropriate ratio or used as it is. .

  The protein phosphatase to be subjected to profiling may be a commercially available reagent, but it is also possible to use one that is already contained or presumed to be contained in a cell-derived extract. For example, phosphorylation of the immobilized substrate can be performed by directly acting on the array with a protein phosphatase reagent or a protein phosphatase already contained in the cell extract in a buffer or cell extract or a mixture of both. . The dephosphorylation conditions vary depending on the type of protein phosphatase, but it is usually about 10 to 40 ° C., preferably about 20 to 40 ° C. for about 5 minutes to 8 hours, preferably about 10 minutes to 5 hours. Thus, the peptide or protein can be dephosphorylated.

  In the present invention, a chelate compound having a specific binding property to a phosphate group is used in order to monitor dephosphorylation of a substrate on the array specifically with high sensitivity. And dephosphorylation is detected by monitoring a mode that the binding amount of a chelate compound falls. In particular, it is preferable to detect by directly or indirectly monitoring how the amount of fluorescence or luminescence is quenched. A chelate compound generally refers to a complex formed by coordination of a polydentate ligand or chelating reagent to a metal ion such as zinc, iron, cobalt, or palladium. Compounds having the property of binding to each other are preferred. As the chelate compound, those modified with a fluorescent substance or a chemiluminescent substance as described above may be used directly. Among these, it is more preferable to use a polyamine zinc complex. More preferably, a dinuclear zinc complex having a polyamine compound as a ligand is used. Furthermore, it is more preferable to use a hexaamine dinuclear zinc (II) complex having a dinuclear zinc (II) complex in the basic structure.

  A typical example of such a compound is 1,3-bis [bis (2-pyridylmethyl) amino] -2-hydroxypropanolate (IUPAC name: 1,3-bis [ bis (2-pyrylmethyl) amino] -2-propanolatodiincinc (II) complex) as a ligand is a dinuclear zinc complex (provided that the hydroxyl group of the propanol skeleton is an alcoholate with two zinc divalent ions) However, the present invention is not particularly limited to this compound.

The complex compound described above can be synthesized using a general chemical synthesis technique, but can also be synthesized using a commercially available compound as a raw material. For example, the compound (Zn ₂ L) represented by the above formula (I) is obtained by the following method using commercially available 1,3-bis [bis (2-pyridylmethyl) amino] -2-hydroxypropane and zinc acetate as raw materials. Can be synthesized. To a ethanol solution (100 ml) of 4.4 mmol of 1,3-bis [bis (2-pyridylmethyl) amino] -2-hydroxypropane was added 10M aqueous sodium hydroxide solution (0.44 ml), and then zinc acetate dihydrate. (9.7 mmol) is added. A brown oil can be obtained by distilling off the solvent under reduced pressure. To this residue was added 10 ml of water and dissolved, then 1M aqueous sodium perchlorate solution (3 equivalents) was added dropwise while heating to 70 ° C., and the precipitated colorless crystals were collected by filtration and dried by heating. (I) two perchlorate acetate ion adduct ^{_{(Zn 2 L-CH 3 COO}} - · 2ClO 4 - · H 2 O) represented by the structural formula can be obtained in high yield. This crystal contains one molecule of crystal water.

  In the present invention, a polyamine zinc complex as described above may be modified with biotin. It is more preferable that the biotin modification is carried out via a linear or branched linker structure. The molecular weight of the linker structure is in the range of 500 to 1000, more preferably 600 to 900, and still more preferably 700 to 900. Specifically, the structure shown in the formula (IV) is exemplified, but not particularly limited. When the molecular weight of the biotin-modified polyamine zinc complex exceeds 1000, the stability is also deteriorated, which is not preferable from the viewpoint of the binding efficiency to phosphoric acid.

  In the above case, the solution concentration of the biotin-modified polyamine zinc complex to be acted on is not particularly limited, but is usually in the range of 1 μM to 10 M, preferably 10 μM to 1 M, more preferably 10 μM to 10 mM. The mode of action on the array is not particularly limited, but the amount of liquid necessary for spreading the biotin-modified polyamine zinc complex solution over the entire array surface may be dropped, or the array may be immersed in the solution. Or you may make it act by making a solution contact on an array surface, sending a solution using a pump. The action temperature may be room temperature or may be incubated at a constant temperature of about 20 to 40 ° C. The action time is preferably about 10 minutes to 2 hours, more preferably about 30 minutes to 1 hour.

  In the present invention, it is preferable that after a biotin-modified polyamine zinc complex is allowed to act, a receptor such as avidin or streptavidin is further allowed to act. It is more preferable to make streptavidin act. The concentration of avidin or streptavidin to be actuated is not particularly limited, but is usually in the range of 1 μM to 10 M, preferably 10 μM to 1 M, more preferably 10 μM to 10 mM. The mode of action on the array is not particularly limited, and is the same as in the case of the biotin-modified polyamine zinc complex. In that case, avidin or streptavidin labeled with a fluorescent substance or a chemiluminescent substance as described above may be used.

  Alternatively, after the avidin or streptavidin is allowed to act, an antibody that recognizes avidin or streptavidin is allowed to act further, so that the detection sensitivity can be further increased. In that case, it is preferable to use those labeled with a fluorescent substance or a chemiluminescent substance as described above. The concentration at which the antibody is allowed to act is not particularly limited, but is preferably about 0.01 to 10 μg / ml, more preferably about 0.1 to μg / ml. As the antibody, either a monoclonal antibody or a polyclonal antibody can be applied, but the monoclonal antibody is more preferable in terms of specificity. The mode of action on the array is also not particularly limited, and is the same as in the case of biotin-modified polyamine zinc complex, avidin or streptavidin.

  Further, a biotin-modified polyamine zinc complex, avidin or streptavidin may be allowed to act sequentially, but a biotin-modified polyamine zinc complex and a complex of avidin or streptavidin previously formed may be allowed to act directly. In this case, as described above, an antibody that recognizes avidin or streptavidin may be allowed to further act. In forming the complex, it is preferable that the biotin-modified polyamine zinc complex and avidin or streptavidin are reacted in a molar ratio of 1: 1 to 4: 1. The reaction product is preferably purified to remove unreacted product, but the reaction product can be applied as it is.

  The application of such a chelating compound is advantageous in that it can be synthesized at a very low cost by the method described above. In addition, it is stable and easy to use in that it can be stored at room temperature, which is advantageous in terms of distribution. Compared with the detection method using antibodies in particular, it acts regardless of the type of amino acid residue to be phosphorylated and is independent of the reaction with the amino acid sequence in the vicinity of the phosphorylated amino acid. Have great advantages.

  Examples of the protein phosphatase include various tyrosine phosphatases or serine / threonine phosphatases. The types of these protein phosphatases are not particularly limited, and can basically be applied to all types of protein phosphatases.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated more concretely, this invention is not specifically limited to an Example.

(Peptide immobilization)
A gold slide (coated content: first layer of chrome 20 mm, second layer of 450 mm) obtained by depositing gold on a slide glass (S1226 manufactured by Matsunami Glass Industry; size: 26 mm × 76 mm × 1 mm), 8-Amino- with a concentration of 1 mM. It was immersed in an ethanol solution of 1-octanethiol and hydrochloride (manufactured by Dojindo) at room temperature for 1.5 hours. In this way, an amino group surface was formed. The gold slide was washed with ethanol and water, dried, and then dissolved in a 0.4 mg / ml SSMCC (Pierce) solution (10 mM phosphate buffer containing 150 mM NaCl; pH 7.4). .) Was spread by dropping 300 μl so as to cover the region on each glass substrate where the peptide was immobilized, and allowed to react at room temperature for 15 minutes. In this way, a maleimide group surface could be formed on the substrate. Thereafter, the glass substrate was washed with ethanol and then water and dried.

  After drying, a synthetic peptide (hereinafter also referred to as a substrate peptide) serving as a substrate is spotted 10 nl on a glass substrate using a spotter (MultiSPRinter (registered trademark) automatic spotter manufactured by Toyobo), and the humidity is maintained. Then, the substrate peptide was immobilized by standing at room temperature for 16 hours. The substrate peptides used were of the amino acid sequences shown in the sequence listing, and FIG. 1 shows the immobilized arrangement of each substrate peptide. No. 1 and 2 (SEQ ID NO: 1) are spots of PTPβ substrate peptide (containing tyrosine phosphotyrosine) which is a tyrosine phosphatase at concentrations of 100 μg / ml and 10 μg / ml, respectively. Nos. 3 and 4 (SEQ ID NO: 2) are PP2A substrate peptides (containing serine phosphatase), which are serine phosphatases, at spots of 100 μg / ml and 10 μg / ml, respectively. 5 and 6 (SEQ ID NO: 3) are spots at a concentration of 100 μg / ml and 10 μg / ml of negative control (containing non-phosphorylated serine) of the substrate peptide of PP2A, respectively. Each peptide has a cysteine residue added to the end, and the substrate peptide is immobilized on the substrate by a coupling reaction between the thiol group of the cysteine residue and the maleimide group on the substrate surface. . The immobilization pattern of the substrate peptide is as shown in FIG. The substrate peptide solution was dissolved in 10 mM phosphate buffer (pH 7.4) containing 150 mM NaCl and 1 mM Tris (2-carboxyethyl) phosphine hydrochloride (manufactured by Tokyo Chemical Industry) to a concentration of 1 mg / ml. Using.

(Blocking of unreacted maleimide groups)
After washing the surface on which the substrate peptide is immobilized with a phosphate buffer, in order to block unreacted maleimide groups, PEG thiol (SUNBRIGHT (registered trademark) MESH-50H manufactured by NOF Corporation) is adjusted to a concentration of 1 mM. It melt | dissolved in the phosphate buffer (20 mM phosphate, 150 mM NaCl; pH 7.2), 300 microliters was poured on the chip | tip, and it was made to react at room temperature for 30 minutes. The molecular weight of PEG thiol used here is 5,000.

(Detection of dephosphorylation)
The array that had been blocked as described above was washed with PBS and water, and dephosphorylated with PP2A. 300 μl of PP2A solution was dropped on the array and dephosphorylated at 30 ° C. The PP2A solution is 50 mM Tris-HCl buffer solution (pH 8.5; 20 mM MgCl ₂ , 1 mM DTT, containing 0.1 mg / ml BSA) so that the concentration of Pase-2A (manufactured by Promega) is 1 unit / μl. Used diluted.

    The array was washed with PBS and water in this order, and then a biotin-modified polyamine zinc complex was allowed to act on it. As the biotin-modified polyamine zinc complex, Phos-tag (registered trademark) BTL-104 (purchased from Nard Laboratory Co., Ltd.) represented by the following formula (IV) was used. Phos-tag (registered trademark) BTL-104 has a concentration of 25 μg / ml, and the lysis solution contains 10 mM HEPES- containing 0.005% Tween 20, 10% (v / v) ethanol, 0.2 M sodium nitrate, 1 mM zinc nitrate. NaOH buffer (pH 7.4) was used. The action was carried out for 1 hour at room temperature.

  Next, fluorescently labeled streptavidin (hereinafter also referred to as SA) was allowed to act. As fluorescently labeled SA, Alexa Fluor532 labeled streptavidin (Molecular Probes) was used at a concentration of 1 μg / ml. Each solution of fluorescently labeled SA was used for the reaction at a concentration of 10 μg / ml for 15 minutes at room temperature. 300 μl of the reaction solution was dropped on the array to spread the solution and reacted at room temperature for 30 minutes.

  The array was washed with PBS and water in that order and dried, and then fluorescence imaging analysis was performed. For the array analysis, a microarray scanner GenePix4200AL (manufactured by Axon Instruments) was used. The results are shown in FIG. In the array without dephosphorylation (0 minutes), fluorescent coloration was observed at the spots (No. 1 to 4) where the phosphorylated peptide was immobilized, and the non-phosphorylated peptide was immobilized. In the spots (No. 5 and 8), no color development is observed. Then, by carrying out the reaction with PP2A, it was confirmed that the fluorescence was quenched with time in the spots (Nos. 3 and 4) where the PP2A substrate was immobilized. In addition, in the spot (No. 3 and 4) to which the substrate peptide of PTPβ is immobilized, there is almost no change in fluorescence. Therefore, it can be said that PP2A has succeeded in detecting the substrate dephosphorylated on-chip.

  According to the method of the present invention, it is possible to analyze various protein phosphatase kinetics very easily and quickly without requiring a special technique. By using a chelate compound, it is inexpensive and easy to handle, and has a great advantage over conventional methods in that it is not affected by the type of phosphorylated amino acid or the amino acid sequence in the vicinity thereof. By using the present invention, it is possible to comprehensively analyze various types of protein phosphatase signals, and effectively profile protein phosphatase kinetics in cells accompanying the introduction of genes with unknown functions or drug administration. be able to. As a result, it is expected to expand into genome drug discovery such as functional analysis from new genes and approaches to new drug discovery, and it will greatly contribute to the industry.

It is a figure which shows the fixed pattern of the peptide on the array produced in the Example. In an Example, it is a figure which shows the result of having performed the array analysis regarding the dephosphorylation in On-chip by PKA.

Claims (10)

  A method for detecting dephosphorylation of a peptide or protein immobilized on a substrate, wherein a chelating compound is allowed to act on the substrate on which the peptide or protein is immobilized, and then phosphorylation is performed using fluorescence or chemiluminescence. A method for detecting protein phosphatase activity, which comprises detecting oxidation.     The method for detecting protein phosphatase activity according to claim 1, wherein the chelating compound is a polyamine zinc complex.     The method for detecting protein phosphatase activity according to claim 1 or 2, wherein the chelating compound is a dinuclear zinc complex having a polyamine compound as a ligand. The method for detecting protein phosphatase activity according to any one of claims 1 to 3, wherein the chelating compound is a compound represented by the formula (I).
    The method for detecting protein phosphatase activity according to any one of claims 1 to 4, wherein the substrate is glass.     The method for detecting protein phosphatase activity according to any one of claims 1 to 5, wherein the surface is gold.     The method for detecting protein phosphatase activity according to any one of claims 1 to 6, wherein a peptide having a cysteine residue added thereto is immobilized.     The method for detecting protein phosphatase activity according to any one of claims 1 to 7, wherein the chelating compound is a compound modified with a fluorescent substance or a chemiluminescent substance.     The protein according to any one of claims 1 to 8, wherein a compound modified with biotin is used as the chelating compound and avidin or streptavidin modified with a fluorescent substance or a chemiluminescent substance is allowed to act on the protein. Method for detecting phosphatase activity.     A protein phosphatase activity according to any one of claims 1 to 9, wherein a complex of a chelating compound modified with biotin and an avidin or streptavidin modified with a fluorescent substance or a chemiluminescent substance is used. Detection method.